The activities of four anticancer alkyllysophospholipids against Leishmania donovani, Trypanosoma cruzi and Trypanosoma brucei

The in-vitro activities of four anticancer alkyllysophospholipids, ET-18-OCH3 (edelfosine), hexadecylphosphocholine (miltefosine), ilmofosine and SRI 62-834, were determined with ED50 values for Leishmania donovani and Trypanosoma cruzi amastigotes between 0.2 and 5.0 microM and for Trypanosoma brucei trypomastigotes between 7.0 and 50.8 microM. In BALB/c mice miltefosine and ilmofosine were the most active compounds with ED50 values of 2.9 and 14.5 mg/kg x 5 doses against L. donovani and a suppressive effect on T. cruzi infections at 30 mg/kg x 5 doses.     

In vitro and in vivo activities of trybizine hydrochloride against various pathogenic trypanosome species

Trybizine hydrochloride [O,O'-bis(4,6-diamino-1,2-dihydro-2, 2-tetramethylene-s-triazine-1-yl)-1,6-hexanediol dihydrochloride] was active in vitro against the sleeping sickness-causing agents Trypanosoma brucei subsp. rhodesiense and T. brucei subsp. gambiense; against a multidrug-resistant organism, T. brucei subsp. brucei; and against animal-pathogenic organisms Trypanosoma evansi, Trypanosoma equiperdum, and Trypanosoma congolense; but not against the intracellular parasites Trypanosoma cruzi and Leishmania donovani. Cytotoxic effects against mammalian cells were observed at approximately 10(6)-fold higher concentrations than those necessary to inhibit T. brucei subsp. rhodesiense. Trybizine hydrochloride was able to eliminate T. brucei subsp. rhodesiense and T. brucei subsp. gambiense in an acute rodent model with four intraperitoneal doses of 0.25 mg kg of body weight-1 or four doses of 1 mg kg-1, respectively, or with four oral doses of 20 mg kg-1. The compound expressed activity against suramin-resistant T. evansi strains in mice. However, these concentrations were not sufficient to cure mice infected with multidrug-resistant T. brucei subsp. brucei. A late-stage rodent model with central nervous system involvement could not be cured, indicating that trybizine may not pass the blood-brain barrier in sufficient quantities.     

Odontogenic tumors. A demographic study of 759 cases in a Chinese population

Hybrid molecules were constructed with either polyclonal antibodies against Trypanosoma cruzi antigens or monoclonal antibody against Trypanosoma brucei brucei low-density lipoprotein (LDL)-receptor conjugated with chlorambucil. Physical-chemical analysis of the hybrid molecule showed four chlorambucil coupling sites in each IgG and a binding constant in the order of 10(4). Maintenance of IgG integrity was indicated by its circular dichroism pattern. Biologic activity of the hybrid molecule was shown by its inhibitory effect on the mobility and proliferation of the parasite. An IgG-chlorambucil conjugate, produced with monoclonal antibody anti-T. b. brucei LDL-receptor, led to the immobilization of the T. cruzi forms, albeit at a much lesser level than that obtained with a mouse polyclonal anti-T. cruzi IgG linked to the drug. Targeting experimental T. cruzi infection with a specific IgG-chlorambucil conjugate resulted in consistent reduction of parasitemia and mortality, thus showing its potential usefulness in controlling the acute form of the disease.     

Neolignans inhibit Trypanosoma cruzi infection of its triatomine insect vector, Rhodnius prolixus

Two neolignans, burchellin and nordihydroguaiaretic acid (NDGA), were toxic only to Trypanosoma cruzi clone Dm28c maintained in brain heart infusion (BHI) medium at a concentration of 100 microg/ml, not 10 microg/ml. When Rhodnius prolixus was fed with epimastigotes of T. cruzi and treated simultaneously with a single dose of burchellin or NDGA at 10 pg/ml of blood meal the number of parasites in the gut decreased. Whereas burchellin was only partially active, NDGA drastically reduced the number of epimastigotes and metacyclic trypomastigotes of T. cruzi in the excreta (urine plus feces). When the insect larvae were pretreated with burchellin or NDGA at 20 days before the infection with T. cruzi a significant reduction in the number of parasites in the gut occurred. However, when both compounds were applied at 20 days after the establishment of T. cruzi infection, although burchellin significantly reduced the gut infection, neither compound could abolish the infection entirely within the subsequent 15 days.     

Pravastatin has cholesterol-lowering independent effects on the artery wall of atherosclerotic monkeys

The amino acid sequence of triosephosphate isomerase from Trypanosoma brucei, Trypanosoma cruzi, and Leishmania mexicana have an identity of 68%. Using the numbering system for the T. brucei enzyme, in their aligned sequences, the T. cruzi and leishmanial enzymes have cysteine residues at positions 14, 40, 117 and 126. T. brucei triosephosphate isomerase has cysteine residues at positions 14, 40 and 126, and a valine residue at position 117. Dithionitrobenzoic acid and methylmethane thiosulfonate inhibited the three enzymes, but T. cruzi triosephosphate isomerase was more than 100-fold more sensitive. The sensitivity of wild type triosephosphate isomerase from T. cruzi and T. brucei to the reagents was equal to that of the Cys117Val and Val117Cys mutant enzymes, respectively. Triosephosphate isomerases that have cysteine residues at positions 40 and 126, but lack a cysteine residue at position 14 are insensitive to methylmethane thiosulfonate. Thus, sulfhydryl reagents act on Cys14. At stoichiometric concentrations, the reagents inhibited the three enzymes as a consequence of structural alterations as measured by binding of 8-anilino-1-napthalenesulfonic acid to previously buried hydrophobic regions. However, the times for half-maximal alterations were 10 min, 15 hours and over 30 hours for T. cruzi, T. brucei and L. mexicana triosephosphate isomerase, respectively. The effect of pH on the action of the sulfhydryl reagents and molecular modeling showed no differences in the solvent accessibility of Cys14. As Cys14 forms part of the dimer interface, the data indicate that, in the three enzymes, barriers of different magnitude hinder the interaction between the sulfhydryl reagents and Cys14. The barrier is lower in T. cruzi triosephosphate isomerase which makes its dimer interface more susceptible for perturbation.     

Structure-activity relationship of new growth inhibitors of Trypanosoma cruzi

Several drugs bearing the 4-phenoxyphenoxy skeleton and other closely related structures were designed, synthesized, and evaluated as antiproliferative agents against Trypanosoma cruzi, the etiologic agent of Chagas' disease. The new class of drugs was envisioned by modifying the nonpolar 4-phenoxyphenoxy moiety replacing selected aromatic protons by different groups via electrophilic aromatic substitution reactions as well as introducing a sulfur atom at the polar extreme. Of the designed compounds, sulfur-containing derivatives were shown to be potent antireplicative agents against T. cruzi. Among these drugs, 4-phenoxyphenoxyethyl thiocyanate (compound 56) proved to be an extremely active growth inhibitor of the epimastigote forms of T. cruzi and displayed an IC50 of 2.2 microM. Under the same assay conditions, this drug was much more active than Nifurtimox, one of the drugs currently in clinical use to control this disease. This thiocyanate derivative was also a very active inhibitor against the intracellular form of the parasite at the nanomolar level. Other sulfur derivatives prepared also exhibited very potent antiproliferative action against T. cruzi. The presence of a sulfur atom at the polar extreme for this family of compounds seems to be very important for biological action because this atom was always associated with high inhibition values. 4-Phenoxyphenoxyethyl thiocyanate presents very good prospective not only as a lead drug but also as a potential chemotherapeutic agent.     

Cloning of a cdc2-related protein kinase from Trypanosoma cruzi that interacts with mammalian cyclins

Two cdc2-related protein kinases (crk), tzcrk3 and tzcrk1, from the protozoan parasite Trypanosoma cruzi were cloned. tzcrk3 encodes a 35 kDa protein sharing 51.5% amino acid identity with human cdc2 and 82% identity with Trypanosoma brucei CRK3. tzcrk1 encodes a 33 kDa protein sharing 52.7% identity with human cdc2 and a high degree of identity (> 78%) with T. brucei CRK1, Leishmania mexicana CRK1 and Trypanosoma congolense CRK1. A recombinant TzCRK1 protein was able to phosphorylate histone HI and retinoblastoma protein. Western blotting using a polyclonal antibody raised against the recombinant TzCRK1 protein showed that the kinase is present in all life cycle stages of the parasite. A PSTAIRE antiserum detected proteins of 32, 33 and 35 kDa, with differential expression in the life cycle of the parasite. Transfection of COS-7 cells with tzcrk1 demonstrated for the first time that a CRK protein can bind mammalian cyclins; TzCRK1 co-immunoprecipitated with cyclins E, D3 and A suggesting a role for this kinase in cell cycle control. These results indicate that T. cruzi might have cyclin homologues that control the activity of the CRK proteins and that a complex mechanism would exist in order to regulate the kinases involved in the cell cycle and the differentiation processes of the parasite.     

Glycosylphosphatidylinositols are required for the development of Trypanosoma cruzi amastigotes

Induction of a glycosylphosphatidylinositol (GPI) deficiency in Trypanosoma cruzi by the heterologous expression of Trypanosoma brucei GPI-phospholipase C (GPI-PLC) results in decreased expression of major surface proteins (N. Garg, R. L. Tarleton, and K. Mensa-Wilmot, J. Biol. Chem. 272:12482-12491, 1997). To further explore the consequences of a GPI deficiency on replication and differentiation of T. cruzi, the in vitro and in vivo behaviors of GPI-PLC-expressing T. cruzi were studied. In comparison to wild-type controls, GPI-deficient T. cruzi epimastigotes exhibited a slight decrease in overall growth potential in culture. In the stationary phase of in vitro growth, GPI-deficient epimastigotes readily converted to metacyclic trypomastigotes and efficiently infected mammalian cells. However, upon conversion to amastigote forms within these host cells, the GPI-deficient parasites exhibited a limited capacity to replicate and subsequently failed to differentiate into trypomastigotes. Mice infected with GPI-deficient parasites showed a substantially lower rate of mortality, decreased tissue parasite burden, and a moderate tissue inflammatory response in comparison to those of mice infected with wild-type parasites. The decreased virulence exhibited by GPI-deficient parasites suggests that inhibition of GPI biosynthesis is a feasible strategy for chemotherapy of infections by T. cruzi and possibly other intracellular protozoan parasites.     

Superantigens but not mitogens are capable of inducing upregulation of E-selectin ligands on human T lymphocytes

A cDNA clone derived from the Trypanosoma cruzi alpha-tubulin gene was isolated and sequenced (Tc alpha tub; L37345). Tc alpha tub revealed an 87.79% and an 85.36% identity with the DNA sequence of T. brucei and Leishmania, respectively. This clone was used to study, by Northern blots, alpha-tubulin gene expression in epimastigotes, cell-cultured derived trypomastigotes and extracellular amastigotes. alpha-tubulin MRNA levels were the same in epimastigotes and trypomastigotes, however, there was a drastic decrease in amastigotes. This clone could be useful to elucidate the regulatory mechanisms of alpha-tubulin gene expression during the differentiation of T. cruzi.     

Protein farnesyltransferase from Trypanosoma brucei. A heterodimer of 61- and 65-kda subunits as a new target for antiparasite therapeutics

We have previously shown that protein prenylation occurs in the Trypanosomatids Trypanosoma brucei (T. brucei), Trypanosoma cruzi, and Leishmania mexicana and that protein farnesyltransferase (PFT) activity can be detected in cytosolic extracts of insect (procyclic) form T. brucei. A PFT that transfers the farnesyl group from farnesyl pyrophosphate to a cysteine that is 4 residues upstream of the C terminus of the Ras GTP-binding protein RAS1-CVIM has now been purified 60,000-fold to near homogeneity from procyclic T. brucei. By screening a mixture of hexapeptides SSCALX (X is 20 different amino acids), it was found that SSCALM binds to T. brucei PFT with sub-micromolar affinity, and affinity chromatography using this peptide was a key step in the purification of this enzyme. On SDS-polyacrylamide gel electrophoresis, the enzyme migrates as a pair of bands with apparent molecular masses of 61 and 65 kDa, and thus its subunits are approximately 30% larger than those of the mammalian homolog. The 61-kDa band was identified as the putative beta-subunit by photoaffinity labeling with a 32P-labeled analog of farnesyl pyrophosphate. Mimetics of the C-terminal tetrapeptide of prenyl acceptors have been previously shown to inhibit mammalian PFT, and these compounds also inhibit T. brucei PFT with affinities in the nanomolar to micromolar range, although the structure-activity relationship is very different for parasite versus mammalian enzyme. Unlike mammalian cells, the growth of bloodstream T. brucei is completely inhibited by low micromolar concentrations of two of the PFT inhibitors, and these compounds also block protein farnesylation in cultured parasites. These compounds also potently block the growth of the intracellular (amastigote) form of T. cruzi grown in fibroblast host cells. The results suggest that protein farnesylation is a target for the development of anti-trypanosomatid chemotherapeutics.     

Concurrence of Graves'' disease and dysplastic cerebral blood vessels of the moyamoya variety

A recent observation that (+)-7-deaza-5'-noraristeromycin (1), as an L-like analogue of aristeromycin, possessed meaningful anti-trypanosomal properties has prompted a search of other 7-deazapurines with similar or improved anti-trypanosomal responses. In that direction a series of pyrazolo[3,4-d]pyrimidines (that is, 8-aza-7-deaza-5'-noraristeromycin derivatives, 2-11) related to 1 have been prepared. These derivatives were evaluated against bloodstream forms of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense grown in vitro. Of these compounds, the parent L-like derivative 2 was less potent (IC50 40-70 microM) than 1 (IC50 0.165-5.3 microM) whereas the D-like analogue 3 was inactive, which is the same trend observed previously with 7-deaza-5'-noraristeromycin. Interestingly, some moderate activity (IC50 12.2-16.8 microM) was seen in the D-like 4'-methyl derivative 7 while its L-like partner was inactive.     

Dihydrolipoamide dehydrogenase in the trypanosoma subgenus, trypanozoon

The enzyme dihydrolipoamide dehydrogenase has been discovered and characterised in four salivarian trypanosomes of the subgenus trypanozoon: Trypanosoma brucei brucei, T. b. gambiense, T. b. rhodesiense, and Trypanosoma evansi. The three T. brucei species, which have insect procyclic forms biochemically distinct from their mammalian bloodstream forms, express dihydrolipoamide dehydrogenase in both cell types, but have higher levels in the procyclic forms. Determination of Michaelis constants for the enzyme from each of the three T. brucei species did not reveal any significant kinetic differences between the bloodstream and procyclic enzymes. On Western blots, antibodies raised against dihydrolipoamide dehydrogenase from the stereorarian trypanosome, Trypanosoma cruzi, cross-react strongly with the dihydrolipoamide dehydrogenase from all three T. brucei species; by this method, the relative molecular masses of their dihydrolipoamide dehydrogenases are indistinguishable. Dihydrolipoamide dehydrogenase was purified from both the bloodstream and the procyclic forms of T. b. brucei, and the N-terminal have been sequenced. These sequences are identical to the derived protein sequence of the cloned gene (Else et al., Eur. J. Biochem. 212 (1993) 423-429), but have a nine amino acid N-terminal truncation, giving an N-terminus equivalent to that of T. cruzi dihydrolipoamide dehydrogenase. The T. b. brucei dihydrolipoamide dehydrogenase gene has been expressed in Escherichia coli and the resultant protein purified; its N-terminus is processed in a similar fashion to that in the trypanosome, but with reduced specificity.     

Mechanism of action of E7010, an orally active sulfonamide antitumor agent: inhibition of mitosis by binding to the colchicine site of tubulin

E7010 (N-[2-[(4-hydroxyphenyl)amino]-3-pyridinyl]-4-methoxybenzenesulfonami de), an orally active sulfonamide antitumor agent that is currently in a Phase I clinical trial, showed rather consistent growth-inhibitory activities against a panel of 26 human tumor cell lines (IC50 = 0.06-0.8 microg/ml), in contrast to vincristine (VCR; IC50 = 0.0002-0.04 microg/ml), 5-fluorouracil (IC50 = 0.2-30 microg/ml), Adriamycin (IC50 = 0.002-0.7 microg/ml), mitomycin C (IC50 = 0.007-3 microg/ml), 1-beta-D-arabinofuranoxylcytosine (IC50 = 0.005 to >30 microg/ml), camptothecin (IC50 = 0.002-0.4 microg/ml), and cisplatin (IC50 = 0.5-20 microg/ml). It caused a dose-dependent increase in the percentage of mitotic cells in parallel with a decrease in cell proliferation, like VCR. It also showed a dose-dependent inhibition of tubulin polymerization, which correlated well with the cell growth-inhibitory activity. 14C-labeled E7010 bound to purified tubulin, and this binding was inhibited by colchicine but not by VCR. However, its binding properties were different from those of colchicine, as well as those of VCR. E7010 was active against two kinds of VCR-resistant P388 cell lines, one of which showed multidrug resistance due to the overexpression of P-glycoprotein (resistant to Taxol), and the other did not show multidrug resistance (sensitive to Taxol). Furthermore, four E7010-resistant P388 cell lines showed no cross-resistance to VCR, a different pattern of resistance to podophyllotoxin, and collateral sensitivity to Taxol. Therefore, E7010 is a novel tubulin-binding agent that has a wider antitumor spectrum than VCR and has different properties from those of VCR or Taxol.     

In vitro antitrypanosomal activity of African plants used in traditional medicine in Uganda to treat sleeping sickness

In Uganda, as in many other African countries, herbal treatment of various diseases is still common. In the present study, 9 plant species collected from Tanzania and Uganda and used by traditional healers in southern-eastern Uganda for the treatment of human African trypanosomiasis (sleeping sickness) were extracted and screened for their in vitro activity against Trypanosoma brucei rhodesiense, one of the two causative agents of sleeping sickness. Eight lipophilic extracts of 5 plants revealed very promising antitrypanosomal activity with IC50 values below 1 microgram/ml; among them were extracts prepared from Albizia gummifera (2), Ehretia amoena (1), Entada abyssinica (2), Securinega virosa (1) and Vernonia subuligera (2). Activity with IC50 values between 1 and 10 micrograms/ml was determined for 15 further extracts. Cytotoxicity of active extracts, tested on a human fibroblast cell line (WI-38), was found to be high, and therefore selectivity indices resulted in less favourable ranges than those for the few commercially available drugs. Nevertheless, the results confirm the potential of ethnobotanically selected plants as remedies against sleeping sickness and call for phytochemical studies.     

Cytotoxic and antiparasitic activity from Annona senegalensis seeds

Several extracts of Annona senegalensis (Annonaceae) seeds were tested for antiparasitic activity against Leishmania major, Leishmania donovani, Trypanosoma brucei brucei, and for cytotoxic activity against KB and VERO cell lines. Fractionation of seeds extracts was mainly guided by means of a biocidal assay against Artemia salina nauplii. The biological activities resulted from extract-isolated acetogenins.     

A pteridine reductase gene ptr1 contiguous to a P-glycoprotein confers resistance to antifolates in Trypanosoma cruzi

We have isolated the pteridine reductase-1 gene (ptr1), from Trypanosoma cruzi (Y strain), located contiguous to the Trypanosoma cruzi P-glycoprotein-2 (tcpgp2). The gene encodes a member of the family of short-chain dehydrogenases, enzymes that are involved in several oxidoreduction reactions. One member of the family, pteridine reductase-1 (PTR1) has been previously described in Leishmania as being involved in antifolate resistance. The ptr1 gene from T. cruzi presents an 828 bp open reading frame, coding for a 276 amino acid protein with a predicted molecular mass of 30 kDa. The deduced amino acid sequence exhibited a remarkable homology with the ptr1 genes of Leishmania major and Leishmania tarentolae. Southern blot analysis using a specific probe indicated that T. cruzi PTR1 is encoded by a single copy gene located in two chromosomes of about 0.9 and 1.2 Mb. Western blot analysis using a polyclonal antiserum against recombinant PTR1 revealed that the protein is only expressed in the epimastigote forms of the parasite; we did not detect the protein either in the amastigote or trypomastigote forms. Purified recombinant PTR1 exhibits a NADPH-dependent pteridine reductase activity comparable with those described in Leishmania. Gene transfection experiments using the pTEX expression vector show that, under the conditions tested, T. cruzi PTR1 is involved in resistance to the methotrexate, aminopterin and trimethoprim antifolates.     

Inhibitory activities of gatifloxacin (AM-1155), a newly developed fluoroquinolone, against bacterial and mammalian type II topoisomerases

We determined the inhibitory activities of gatifloxacin against Staphylococcus aureus topoisomerase IV, Escherichia coli DNA gyrase, and HeLa cell topoisomerase II and compared them with those of several quinolones. The inhibitory activities of quinolones against these type II topoisomerases significantly correlated with their antibacterial activities or cytotoxicities (correlation coefficient [r] = 0.926 for S. aureus, r = 0.972 for E. coli, and r = 0.648 for HeLa cells). Gatifloxacin possessed potent inhibitory activities against bacterial type II topoisomerases (50% inhibitory concentration [IC50] = 13.8 microg/ml for S. aureus topoisomerase IV; IC50 = 0.109 microg/ml for E. coli DNA gyrase) but the lowest activity against HeLa cell topoisomerase II (IC50 = 265 microg/ml) among the quinolones tested. There was also a significant correlation between the inhibitory activities of quinolones against S. aureus topoisomerase IV and those against E. coli DNA gyrase (r = 0.969). However, the inhibitory activity against HeLa cell topoisomerase II did not correlate with that against either bacterial enzyme. The IC50 of gatifloxacin for HeLa cell topoisomerase II was 19 and was more than 2,400 times higher than that for S. aureus topoisomerase IV and that for E. coli DNA gyrase. These ratios were higher than those for other quinolones, indicating that gatifloxacin possesses a higher selectivity for bacterial type II topoisomerases.     

(+)-7-Deaza-5''-noraristeromycin as an anti-trypanosomal agent

The (+)-enantiomer of 7-deaza-5'-noraristeromycin (4) has been found to show IC50 values ranging from 0.16 to 5.3 microM against four strains of African trypanosomes, one Trypanosoma brucei brucei isolate, and several clinical isolates of Trypanosoma brucei rhodesiense (agent of east African sleeping sickness), including a multidrug resistant clone of one isolate. While this compound was originally designed to inhibit S-adenosyl-L-homocysteine hydrolase, it has been found to have no effect on this enzyme.     

A novel antiprotozoal aminosteroid from Saracha punctata

A new aminosteroid, 3beta-amino-22,26-epiminocholest-5-ene named sarachine (1), and two known flavonoids, eriodictyol (2) and 7-O-beta-D-glucopyranosyl-eriodictyol (3), were isolated from the leaves of Saracha punctata. The alkaloid was found to inhibit the growth of Leishmania braziliensis promastigotes (100% at 25 microM) and of Trypanosoma cruzi epimastigotes in culture (50% at 25 microM) and showed a strong in vitro antiplasmodial activity with an IC50 of 25 nM.